Therapeutic compounds and methods

ABSTRACT

The present invention provides compounds having a cyclopentabenzofuran core and the use of such compounds in therapy as well as compositions comprising said compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of PCT/AU01/00810filed Jul. 5, 2001.

FIELD OF THE INVENTION

The present invention relates generally to compounds having acyclopentabenzofuran core. More particularly, the present inventionrelates to cyclopentabenzofuran compounds having a bulky substituent atthe 6-oxy position, such as where the cyclopentabenzofuran core issubstituted by a dioxanyloxy moiety. The invention also relates to theuse of these compounds in therapy and compositions comprising saidcompounds.

BACKGROUND

Aglaia is a large genus of the family Meliaceac comprising over 100(mostly woody) species in Indo-Malaysia and the Western Pacific region.Uses include treatment of fever, fractures, parturition andinflammation. Extracts are also used as bactericides, insecticides, inperfumery, as an astringent, tonic, a refrigerant (Dr Duke'sPhytochemical and Ethnobotanical Databases) and for the treatment ofabdominal tumours (Pannel, et al, 1992, Kew Bull., (16) 273-283).

More recently, a number of 1H-cyclopenta[b]benzofuran lignans have beenisolated from Aglaia species (see for example, WO97/08161; JP 97171356;Ohse, et al, J Nat Prod, 1996, 59(7):650-52; Lee et al, Chem. Biol.Interact., 1998, 115(3):215-28; Wu et al, J. Nat. Prod., 1997,60(6):606-08; Bohnenstengel et al, Z. Naturforsch, 1999, 54c (12):55-60and Bohnenstengel et al, Z. Naturforsch, 1999, 54c (12):1075-83, Xu, Y.J., et al, 2000, J. Nat. Prod., 63, 4732-76, the entire contents ofwhich are incorporated herein by reference). A number of these compoundshave also been noted for their insecticidal activity (Janpraseri, et al,1993, Phytochemistry, 32 (1): 67-69; Ishibashi et al, 1993,Phytochemistry, 32 (2): 307-310; Hiort, et al, 1999, J. Nat. Prod., 62(12): 1632-1635). Insecticidal compounds with a closely related corestructure were isolated from Aglaia roxburghiana and are described in WO9604284 for use as active ingredients in agrochemical formulations.

New compounds (Compounds A and B), as described herein) have now beenisolated from Aglaia leptantha, Miq. (Meliaceae) which uniquely possessa dioxanyloxy group at the 6-position of the cyclopenta[b]benzofurancore. Compounds A and B have been shown to exhibit potent cytotoxic andcytostatic effects on cancer cell growth and viability and thus thecompounds of the invention and derivatives thereof, may be useful astherapeutic agents in the treatment of cancer and cancerous conditionsor other diseases associated with cellular hyperproliferation.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

In a first aspect, the invention relates to compounds of Formula (I) ora salt or prodrug thereof.

wherein

each R⁴-R¹⁰ is independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted acyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted cycloalkylalkyl, optionally substituted arylacyl,optionally substituted cycloalkylacyl and a C-1 linked saccharide;

X is OR⁸ or NR⁹R¹⁰;

R¹¹ and R¹² are preferably each independently hydrogen or,alternatively, OR⁴ and R¹¹, and/or OR⁵ and R¹² together form amethylenedioxy group; and

Y is selected from the group consisting of optionally substitutedphenyl, optionally substituted benzyl, optionally substituted benzoyl,optionally substituted C₃-C₈ cycloalkyl, (preferably optionallysubstituted C₅-C₆ cycloalkyl) optionally substituted CH₂-(C₃-C₈cycloalkyl) (preferably optionally substituted CH₂-(C₅-C₆ cycloalkyl),optionally substituted 5-6 membered heterocyclyl, and optionallysubstituted CH₂-(5-6 membered heterocyclyl).

In a preferred embodiment, the invention relates to compounds (includingsteroisomers within the dioxanyl group) of formula (i) or a salt orprodrug thereof.

wherein

and each R¹-R¹⁰ is independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted acyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted cycloalkylalkyl, optionally substituted arylacyl,optionally substituted cycloalkylacyl and a C-1 linked saccharide; and

X is OR⁸ or NR⁹R¹⁰);

R¹¹ and R¹² are each independently hydrogen or, OR⁴ and R¹¹, and/or OR⁵and R¹² together form a methylenedioxy group. In one preferredembodiment, R¹¹ and R¹² are both hydrogen.

In one preferred embodiment, compounds of the present invention have theFormula (ii):

or a salt or prodrug thereof.

Formula (ii) has 4 chiral centres in the dioxanyl moiety. Two isomers(isomeric in the dioxanyl group) of Formula (ii) have now beenisolated—Compounds A and B as described in Example 1.

In another aspect, the invention provides a composition comprising acompound of Formula (I), such as Formula (i), or a salt or prodrugthereof, together with a pharmaceutically acceptable carrier, excipientor diluent.

In still a further aspect, the present invention provides a method forthe treatment of cancer or a cancerous condition or a disease state orcondition associated with cellular hyperproliferation comprising theadministration of a treatment effective amount of a compound of Formula(I), such as Formula (i), or a salt, derivative or prodrug thereof, to asubject in need thereof. Some particular cancerous conditions which maybe treated by the compounds of the invention may include lung, prostate,colon, brain, melanoma, ovarian, renal and breast tumours and leukemia.Disease states or conditions associated with cellular hyperproliferationwhich may be treated by compounds of the invention may includeatherosclerosis, restinosis, rheumatoid arthritis, osteoarthritis,inflammatory arthritis, psoriasis, peridontal disease or virally inducedcellular hyperproliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Compound A Promotes Differentiation of THP-1 leukemic cells.

THP-1 cells were cultured for 4 days in the presence or absence of 10 nMCompound A as indicated. Where shown cells were also treated with IFNγ(100 ng/ml) (3 days) or with PMA (0.1 μM) (4 days) in the presence orabsence of Compound A. Images are of cells visualised by phase contrastmicroscopy (magnification×200).

FIG. 2: Effects of Compound A on Cell Cycle Progression and Viability ofTHP-1 Cells.

THP-1 cells were cultured for 2 days with the indicated concentration ofCompound A or 1000 nM paclitaxel then collected and fixed in 70% ethanolprior to staining with propidium iodide and DNA content determined byflow cytometry. The numbers indicate the % of cells in the various cellcycle phases relative to all cells with ≧2N DNA content and also the %dead cells (ie. subdiploid≦2N cells) to the left of the marker (thevertical line) that arose during the culture period.

FIG. 3: Effects of Compound A on the proliferation of A549 cells.

A549 cells were seeded at ˜10,000 cells/well and cultured in thepresence of the indicted concentrations of Compound A or paclitaxel.Cells were collected and the viable cell number determined byhaemocytometer counting of trypan blue stained cells at the varioustimes. The results are the averages ±SEM of triplicate cultures.

FIG. 4: Effects of Compound A on Cell Cycle Progression and Viability ofA549 Cells.

A549 cells were cultured for 6 days with the indicated concentration ofCompound A or 1 μM paclitaxel then collected and fixed in 70% ethanolprior to staining with propidium iodide and DNA content determined byflow cytometry. The numbers indicate the % of cells in the various cellcycle phases relative to all cells with ≧2N DNA content and also the %dead cells (ie. subdiploid≦2N cells) to the left of the marker thatarose during the culture period.

FIG. 5: Compounds A and B Induce G2/M Phase Accumulation of K362Leukemic Cells

K562 cells were cultured for 3 days with the indicated concentration ofCompounds A or B then collected and fixed in 70% ethanol prior tostaining with propidium iodide and DNA content determined by flowcytometry. The numbers indicate the % of cells in G0/G1, S and G2/Mphases of the cell cycle respectively relative to all cells with ≧2N DNAcontent.

FIG. 6: Cytostatic Effects of Compound A on A549 Cells are Reversible

A549 cells were seeded at ˜10,000 cells/well and cultured in thepresence of the indicted concentrations of Compound A or paclitaxel andthe viable cell numbers determined by haemocytometer counting of trypanblue stained cells at the various times. On day 5 some of the cells werewashed, resuspended in fresh medium lacking the various treatments andcultured for another 4 days prior to counting.

FIG. 7: Compound A Inhibits Camptothecin- and Paclitaxel-InducedCytotoxicity of A549 Cells

A549 cells in 96 well plates were cultured for 3 days in the presence orabsence of 10 nM Compound A together with the indicated concentrationsof (A) camptothecin or (B) paclitaxel. Loss of membrane integrity wasthen assessed by the addition of the fluorescent DNA-binding dye YOYO-1and the increased fluorescence accompanying cell death measured using afluorescent plate reader.

FIG. 8: Compound A Inhibits Cell Cycle Arrest and Cell Death Induced byAnti-Cancer Agents but not by Staurosporine

A549 cells in 6 well plates were cultured for 3 days in the presence orabsence of 10 nM Compound A together with 0.1 μM camptothecin, 10 μMvinblastin, 1 μM paclitaxel or 1 μM staurosporine as indicated. Thecells were then collected and fixed in 70% ethanol prior to stainingwith propidium iodide and DNA content determined by flow cytometry. Thenumbers indicate the % of cells in the various cell cycle phasesrelative to all cells with ≧2N DNA content and also the % dead cells(ie. subdiploid ≦2N cells) to the left of the dotted marker that aroseduring the culture period.

FIG. 9: Compound A Does not Induce Senescence-Associated β-GalactosidaseActivity in A549 Cells

A549 cells were seeded at 10,000 cell/well in 6 well plates in thepresence or absence of varying concentrations of Compound A (10-50 nM)or 250 nM doxorubicin for 10 days prior to their processing and stainingovernight for senescence-associated β-galactosidase activity asdescribed previously (Dimri et al., 1995, Proc Natl Acad Sci USA 199592(20):9363-7). For Compound A only the 10 nM treatment is shown butthere was no detectable SA-β gal activity at any other concentrationstested. PC, phase contrast microscopy. BF, bright field microscopyMagnification×200.

FIG. 10: Compound A Inhibits Growth of Human Tumour Cells in a MouseXenograft Model

Athymic Balb/c nude mice (Rygard and Povisen, 1969, Acta PatholMicrobiol Scand, 77: 758) were inoculated subcutaneously in the dorsalflank with 2×10⁶ PC3 cells. Compound A was administered (3 mg/kg) aftereight days when the tumours became palpable by intraperitoneal injectionthree times a week. Compound A was first solubilized in ethanol thenmixed 1:1 with cremaphore and diluted in saline for injection. Controlanimals were treated in an analogous manner with the same vehicle butlacking Compound A. (A) Effect of Compound A on mean tumour volume.Tumour volumes were measured using a micrometer caliper at the indicatedtimes. The data represents mean tumour volume ±SEM (B) Effect ofCompound A on mean tumour mass. At the end of the experiment (29 dayspost inoculation of PC3 cells) the mice were sacrificed, the tumoursexcised and then weighed. The data represents mean tumour weight ±SEM.

DETAILED DESCRIPTION OF THE INVENTION

Cyclopenta[b]benzofurans previously reported carry a methoxy group orsimilar small substituent (Greger et al, 2001, Phytochemistry, 57, (1);57-64) at the 6- or 8-positions. In contrast, the compounds of thepresent invention carry a sterically bulky group at the 6-oxy-position,in particular, a dioxanyl group. The dioxanyl group of Formula (ii)(depicted below as sub-Formula (a)) has not previously been reportedfrom a natural source. Without intending to limit the invention bytheory, it is believed that the presence at the 6-oxy-position of asterically bulky group, ie spatially larger than a methoxy group, mayconfer both cytotoxic and cytostatic properties on the compounds havinga cyclopenta[b]benzofuran core.

The invention includes within its scope pharmaceutically acceptablesalts, derivatives, or prodrugs of compounds of Formula (I),particularly of Formula (i), such as Compounds A and B.

The term “salt, or prodrug” includes any pharmaceutically acceptablesalt, ester, glycoside, solvate, hydrate or any other compound which,upon administration to the recipient subject is capable of providing(directly or indirectly, for example, by chemical or in vivo enzymaticor hydrolytic degradation) a compound of the invention as describedherein.

Suitable pharmaceutically acceptable salts include salts ofpharmaceutically acceptable inorganic acids such as hydrochloric,sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, andhydrobromic acids, or salts of pharmaceutically acceptable organic acidssuch as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic,fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic,phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic,salicyclic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic,oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Basesalts include, but are not limited to those formed with pharmaceuticallyacceptable cations, such as sodium, potassium, lithium, calcium,magnesium, ammonium and alkylammonium.

The preparation of salts can be carried out by methods known in the art.It will also be appreciated that non-pharmaceutically acceptable saltsalso fall within the scope of the invention, since these may be usefulas intermediates in the preparation of pharmaceutically acceptablesalts.

The compounds of the invention may be in crystalline form or as asolvate (e.g., hydrates). Methods of solvation will be known to thoseskilled in the art.

Prodrugs of compounds of formula (I) are also within the scope of theinvention. The term “prodrug” includes derivatives that are convertedits vivo to the compounds of the invention and include for example,ester (eg acetate) and glycoside derivatives of free hydroxy groups,which may undergo in vivo degradation to release a compound of theinvention. Other suitable prodrugs may include esters of free carboxylicacid groups. The preparation of suitable prodrugs is further describedin Design of Prodrugs, H. Bundgaard, Elseveir, 1985, the contents ofwhich is incorporated by reference.

It will also be recognised that certain Y groups of Formula (I), inparticular the dioxanyl groups of compounds as depicted in Formula (i)and (ii), may possess asymmetric centres and are therefore capable ofexisting in more than one stereoisomeric form. The invention thus alsorelates to compounds in substantially pure isomeric form at one or moreasymmetric (chiral) centres eg., greater than about 90% ee, such asabout 95% or 97% ee, preferably greater than 99% ee, as well asmixtures, including racemic mixtures, thereof. Such isomers may beresolved by conventional methods, eg, chromatography, or use of aresolving agent. The present invention thus provides Compounds A and B.

As used herein, the term “alkyl”, when used alone or in compound wordssuch as “arylalkyl” refers to a straight chain, branched or cyclichydrocarbon group, preferably C₁₋₂₀, such as C₁₋₁₀. The term “C₁-C₆alkyl” refers to a straight chain, branched or cyclic alkyl group of 1to 6 carbon atoms. Examples of “C₁₋₆ alkyl” include methyl, ethyl,iso-propyl, n-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, isopentyl,2,2-dimethypropyl, n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl,3-methylpentyl and 2,3-dimethylbutyl. Examples of cyclic C₁₋₆ alkylinclude cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Otherexamples of alkyl include: heptyl, 5-methylhexyl, 1-methylhexyl,2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl,1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethyl-pentyl,1,2,3-trimethylbutyl, 1,1,-trimethylbutyl, 1,1,3-trimethylbutyl, octyl,6-methylheptyl, 1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-,3-, 4-, 5-, 6- or 7-methyl-octyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-,2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-ethylnonyl, 1-, 2-, 3-, 4- or5-propylocytl, 1-, 2- or 3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-,3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-,7- or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or4-butyloctyl, 1-2-pentylheptyl and the like. An alkyl group may beoptionally substituted by one or more optional substituents as hereindefined. Optionally, the straight, branched or cyclic hydrocarbon group(having at least 2 carbon atoms) may contain one, two or more degrees ofunsaturation so as to form an alkenyl or alkynyl group, preferably aC₂₋₂₀ alkenyl, more preferably a C₂₋₆ alkenyl, or a C₂₋₂₀ alkynyl, morepreferably a C₂₋₆ alkynyl. Examples thereof include a hydrocarbonresidue containing one or two or more double bonds, or one or two ormore triple bonds. Thus, “alkyl” is taken to include alkenyl andalkynyl.

The term “aryl”, when used alone or in compound words such as“arylalkyl”, denotes single, polynuclear, conjugated or fused residuesof aromatic hydrocarbons or aromatic heterocyclic (heteroaryl) ringsystems, wherein one or more carbon atoms of a cyclic hydrocarbonresidue is substituted with a heteroatom to provide an aromatic residue.Where two or more carbon atoms are replaced, this may be by two or moreof the same heteroatom or by different heteroatoms. Suitable heteroatomsinclude O, N, S and Se.

Examples of “aryl” include phenyl, biphenyl, terphenyl, quaterphenyl,naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl,benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl,idenyl, azulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl,thienyl, furyl, pyrrolyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl,thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl,benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benoxazolyl,benzothiazolyl and the like. Preferred hydrocarbon aryl groups includephenyl and naphthyl. Preferred heterocyclic aryl groups include pyridyl,thienyl, furyl, pyrrolyl An aryl group may be optionally substituted byone or more optional substitutents as herein defined.

The term “acyl” refers to a group —C(O)—R wherein R is any carboncontaining moiety such as an optionally alkyl or substituted aryl group.Examples of acyl include straight chain or branched alkanoyl such as,acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl,2,2-dimethylpropanyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl,undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl,hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and icosanoyl;cycloalkylcarbonyl, such as cyclopropylcarbonyl cyclobutylcarbonyl,cyclopentylcarbonyl and cyclohexylcarbonyl; aroyl such as benzoyl,toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e.g.phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutylyl,phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e.g.naphthylacetyl, naphthylpropanoyl and naphthylbutanoyl]. Since the Rgroup may be optionally substituted as described above, “acyl” is takento refer to optionally substituted acyl.

Optional substituents for alkyl, aryl or acyl include halo (bromo,fluoro, chloro, iodo), hydroxy, C₁₋₆alkyl (eg methyl, ethyl, propyl (n-and i- isomers)), C₁₋₆alkoxy (eg methoxy, ethoxy, propoxy (n- and i-isomers), butoxy (n-, sec- and t-isomers), nitro, amino, C₁₋₆alkylamino(eg methyl amino, ethyl amino, propyl (n- and i- isomers)amino),C₁₋₆dialkylamino (eg dimethylamino, diethylamino, diisopropylamino),halomethyl (eg trifluoromethyl, tribromomethyl, trichloromethyl),halomethoxy (eg trifluoromethoxy, tribromomethoxy, trichloromethoxy) andacetyl. Furthermore, optional substituents for Y (phenyl, benzyl,benzoyl, C₃-C₈ cycloalkyl, CH₂-(C₃-C₈ cycloalkyl), 5-6membered-heterocyclyl and CH₂-(5-6 membered-heterocyclyl)) include, aswell as the substituents above, alkyl substituted with one or more ofhydroxy C₁₋₆alkyloxy, C₁₋₆acyloxy, aryloxy, arylC₁₋₆alkyloxy,C₁₋₆cycloalkylC₁₋₆alkyloxy, arylC₁₋₆acyloxy, C₁₋₆cycloalkylC₁₋₆acyloxyand C1-linked saccharidoxy.

The term “arylalkyl” and “cycloalkylalkyl” refer to an alkyl group(preferably straight chain) substituted (preferably terminally) by anaryl and a cycloalkyl group, respectively. Similarly, the terms“arylacyl” and “cycloalkylacyl” refer to an acyl group (preferably whereR is straight chain alkyl) substituted (for example, terminallysubstituted) by an aryl and a cycloalkyl group, respectively

Preferred C-1 linked saccharides are a furanose or pyranose saccharide(sugar) substituent which is linked to the backbone structure shown inFormula (I) through the saccharides's 1-carbon (conventional chemicalnumbering) to form an acetal at any one of positions R₁, R₂, R₃, R₄, R₅,R₆, or R₇ or an ester linkage at the R₈ or an amide at R₉ or R₁₀position. Exemplary saccharide groups include reducing sugars such asglucose, ribose, arabinose, xylose, mannose and galactoses, each beinglinked to an oxygen atom of the structure of Formula (I) through the C-1carbon of the saccharide group.

A 5-6 membered heterocyclyl group includes aromatic 5-6-memberedheterocyclic aryl groups (heteroaryl) as described above and nonaromatic 5-6-membered heterocyclic groups containing one or moreheteroatoms (preferably 1 or 2) independently selected from O, N, S andSe. Examples thereof include dioxanyl, pyranyl, tetrahydrofuranyl,piperidyl, morpholino, piperazinyl, thiomorpholino and saccharides, forexample, those described above.

In one embodiment of Formula (I) or Formula (i) of the invention, eachof R⁴-R⁷ and R¹-R⁷ respectively may independently be selected from thegroup consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl,n-butyl, sec-butyl, t-butyl, cyclopropylmethyl (or cyclopropylethyl),cyclobutylmethyl (or -ethyl), cyclopentylmethyl (or -ethyl),cyclohexylmethyl (or -ethyl), phenyl, benzyl, acetyl and C-1 linkedsaccharide.

In another embodiment of Formula (I), (i) or (ii) of the invention, R⁸of X═OR⁸ is selected from the group of hydrogen, C₁₋₆ alkyl, phenyl,benzyl and C-1 linked saccharide.

In another embodiment of Formula (I), (i) or (ii) of the invention R⁹and R¹⁰ of X═NR⁹R¹⁰ are independently selected from hydrogen, C₁₋₆alkyl, phenyl or benzyl.

The derivatisation of hydroxy groups of Compounds A and B to formcompounds of Formula (i), (ie where any one of R¹-R⁷ is not hydrogen)can be carried out by methods known in the art for alkylating, arylatingor acylating hydroxy groups, for example as described in ProtectiveGroups in Organic Synthesis T. W. Greene and P. G. M. Wutz, (1999) WileyInterscience, New York, and Advanced Organic Chemisty, J. March, (4^(th)Edition), Wiley-InterScience (the entire contents of which areincorporated herein by reference). For example, hydroxy groups can bealkylated using alkyl halides such as methyl iodide or dialkyl sulfatessuch as dimethyl and diethyl sulfate. Acylation can be effected bytreatment with appropriate carboxylic acids, acid halides or acidanhydrides in the presence of a base or coupling agent. Benzylation maybe effected by treatment with a benzyl halide compound such as benzylbromide, chloride or iodide. De-esterification of the methyl ester canbe effected by treatment of the ester with aqueous base. Esterificationof a carboxylic acid can be achieved by conventional means includingtreatment with an appropriate alcohol in the presence of acid, ortreatment with alkyl sulfates or alkyl halides.

Glycosidic formation may be effected chemically, eg by reacting thestarting compound with a protected sugar compound in which C-1 has beenactivated by halogenation for coupling with the hydroxyl or carboxylgroups and the sugar hydroxyls have been blocked by protecting groups.Alternatively, glycoside formation may be effected enzymatically usingan appropriate glycosyltransferase such as UDP-galactose dependentgalactocyltransferase and UDP-glucose dependent glycocyltransferase(SIGMA).

The skilled person will recognise that in order to selectively installany one or more of the R¹-R¹⁰ groups as defined herein (eg where R¹-R⁷are not hydrogen), this may require the judicious protection and/ordeprotection, of one or more of the oxy and/or carboxy groups. Selectivederivatisation of one or more hydroxy or carboxy groups may be achievedvia conventional techniques by the use of protecting groups withdifferent degrees of stability under appropriate conditions.

Methods for the conversion of a carboxylic acid or ester group; ie.where X is OR⁸ to an amide (X is NR⁹R¹⁰) are known to the skilled personand may include treatment of a carboxylic acid with an appropriate aminein the presence of a coupling reagent such as DCC or treatment of anacid halide with the appropriate amine. Other methods which may besuitable are described in Larock, R. E, Comprehensive OrganicTransformations pp 963-995, VCH Publishers (1989).

As used herein, the term “protecting group”, refers to an introducedfunctionality which may temporarily render a particular functionalgroup, eg hydroxy or carboxylic acid, inactive under certain conditionsin which the group might otherwise be reactive. Suitable protectinggroups are known to those skilled in the art, for example as describedin Protective Groups in Organic Synthesis (supra). Suitable protectinggroups for hydroxy include alkyl, (such as C₁-C₆alkyl), acyl (such asC(O)C₁-C₆alkyl, benzoyl and the like), benzyl, and silyl groups (such astrimethylsilyl, t-butyldimethyl silyl, t-butyldiphlenylsilyl and thelike). Other suitable groups for hydroxy substituents and a carboxysubstituent (acid, amide etc) can be found within Greene supra. Thestability of various groups under certain conditions is understood bythe skilled person and is further exemplified in Protective Groups inOrganic Synthesis (supra).

It will be appreciated that these protected compounds may be useful asintermediates in the preparation of certain compounds of Formula (I) andtherefore, these also form a further aspect of the invention.

It will also be recognised that some groups, eg alkyl, acyl orarylalkyl, (such as methyl, ethyl, propyl, acetyl, benzyl etc) may serveas either a temporary protecting group or as a non-hydrogen R¹-R⁸ groupin Formula (I).

The dioxanyl group may be cleaved from the 6-oxy position of thecyclopentabenzofuran core using known methods to afford a dioxanecompound. The resulting dioxane compound could be used to substituteother compounds, such as oxy-substituted compounds, including thecorresponding 6-oxy position, or other oxy positions, on othercyclopentabenzofuran compounds such as those described in the referencesherein.

It will also be understood that cyclopentabenzofuran compounds, having amethoxy substituent at the 6-position, such as those described in thereferences cited herein (incorporated herein by reference) eg ReferenceCompounds 1-3 (as described in Example 4), can, where appropriate be6-demethylated, and the resulting 6-hydroxy group reacted with asuitable Y precursor to form an 6-OY group. Methods therefor are knownin the art, for example, one method may involve reacting the 6-OH groupwith a Y-halogen compound where halogen includes Cl, Br and I.Alternatively, access to the cyclopentabenzofuran core, incorportationof the Y group can be achieved via synthetic methods analogous to thatdescribed in Trost et al, J. Am. Chem. Soc., 1990, 112, 9022-9024. Such6-OY compounds form a further aspect of the invention.

In some preferred embodiments of the present invention, one or more ofthe following definitions apply:

R¹ and R² are both hydrogen.

R¹ and R² are hydrogen, and R³ is methyl.

at least one of R³-R⁵ is methyl, ethyl or propyl, preferably methyl.

at least two of R³-R⁵ are methyl, ethyl or propyl, preferably methyl.

all of R³-R⁵ are methyl, ethyl or propyl, preferably methyl.

R⁶ and R⁷ are both hydrogen.

at least one of R¹¹ and R¹² is hydrogen, preferably R¹¹ and R¹² are bothhydrogen.

X is OR⁸ where R⁸ is selected from hydrogen, methyl, ethyl or propyl,preferably, methyl.

X is NR⁹R¹⁰ where R⁹ and R¹⁰ are both hydrogen or methyl; or R⁹ and R¹⁰are different but at least one of R⁹ or R¹⁰ is hydrogen and the other isC₁₋₆ alkyl, such as methyl, ethyl or propyl.

Y is an optionally substituted 5-6 membered heterocyclyl group or anoptionally substituted C₅-C₆ cycloalkyl group.

Particularly preferred forms of Formula (ii) are Compounds A and B.

The compounds of the invention may have use in the treatment ofcancerous conditions, or other conditions associated with cellularhyperproliferation, in a subject. Subjects which may be treated by thecompounds of the invention include mammals, for example, humans,primates, livestock animals (eg. sheep, cows, horses, goats, pigs),companion animals, (eg. dogs, cats, rabbits, guinea pigs), laboratorytest animals, (eg, rats, mice, guinea pigs, dogs, rabbits, primates) orcaptured wild animals. Most preferably, humans are the subjects to betreated.

As used herein the term “treatment” is intended to include theprevention, slowing, interruption or halting of the growth of a cancer,tumour or hyperproliferative cell, or a reduction in the number oftargeted cells (or size of the growth mass) or the total destruction ofsaid cell, wherein said cells are cancer, tumour or hyperproliferativecells.

Cancerous conditions which may be treated by the compounds of thepresent invention include conditions wherein the cancers or tumours maybe simple (monoclonal, ie composed of a single neoplastic cell type),mixed (polyclonal, ie. composed of more than one neoplastic cell type)or compound (ie. composed of more than one neoplastic cell type andderived from more than one germ layer) and may include benign andmalignant neoplasia/hyperplasia. Some examples of cancerous conditionswhich may be treated by the present invention include leukemia andbreast, colon, bladder, pancreatic, endometrial, head and neck,mesothelioma, myeloma, oesophagal/oral, testicular, thyroid, uterine,prostate, renal, lung, ovarian, cervical brain, skin, liver, bone, boweland stomach cancers, sarcomas, tumours and melanomas. Examples of benignhyperplasias include those of vascular (eg hemangioma), prostate, renal,adrenal, hepatic, colon (eg colonic crypt), parathyroid gland and othertissues.

As the compounds of the invention may have cytostatic as well ascytotoxic properties, they may also have potential use as therapeuticagents in the suppression of the growth of target populations of cellsother than cancer or tumour cells, for example disease states orconditions associated with cellular hyperproliferation. Such conditionsmay include atherosclerosis and restinosis (neointimal hyperplasia) andhyperproliferation due to or accompanying an inflammatory response, egarthritis, (including rheumatoid arthritis, osteoarthritis andinflammatory arthritis), psoriasis and periodontal disease, or cellularhyperproliferation due to the viral infection of cells such as humanpapilloma virus.

The compounds of the invention, eg Compounds A and B, may be used intherapy in conjunction with other therapeutic compounds, such asanti-cancer compounds, including paclitaxel, camptothecin, vinblastinand doxorubicin.

Thus, another aspect of the invention relates to a method for thetreatment of cancer or a cancerous condition comprising theadministration of an effective amount of a compound of Formula (I) and afurther therapeutic agent to a subject in need thereof, and the use ofsaid compound in the manufacture of a medicament for use in conjunctionwith other therapeutic agents.

The compounds of the invention and the further therapeutic agent may beadministered simultaneously, as a single composition or as discretecompositions, or may be administered separately, ie, one after the otherat suitable intervals as determined by the attending physician. Thus,the invention also provides a kit comprising a compound of Formula (I)together with a further therapeutic agent.

As used herein, the term “effective amount” of a compound relates to anamount of compound which, when administered according to a desireddosing regimen, provides the desired therapeutic activity. Dosing mayoccur at intervals of minutes, hours, days, weeks, months or years orcontinuously over any one of these periods. Suitable dosages lie withinthe range of about 0.1 ng per kg of body weight to 1 g per kg of bodyweight per dosage. The dosage is preferably in the range of 1 μg to 1 gper kg of body weight per dosage, such as is in the range of 1 mg to 1 gper kg of body weight per dosage. In one embodiment, the dosage is inthe range of 1 mg to 500 mg per kg of body weight per dosage. In anotherembodiment, the dosage is in the range of 1 mg to 250 mg per kg of bodyweight per dosage. In yet another embodiment, the dosage is in the rangeof 1 μg to 100 mg per kg of body weight per dosage, such as up to 50 mgper kg body weight per dosage. The dosing regime for each subject may bedependent upon the age, weight, health and medical history of thesubject and the extent and progress of the condition to be treated, andcan be determined by the attending physician.

The active ingredient may be administered in a single dose or a seriesof doses. While it is possible for the active ingredient to beadministered alone, it is preferable to present it as a composition,preferably as a pharmaceutical composition.

The carrier must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the composition and notinjurious to the subject. Compositions include those suitable for oral,rectal, nasal, topical (including buccal and sublingual), vaginal orparental (including subcutaneous, intramuscular, intravenous andintradermal) administration. The compositions may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. Such methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, sachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules optionally mixed with abinder (e.g inert diluent, preservative disintegrant such as sodiumstarch glycolate, cross-linked polyvinyl pyrrolidone, cross-linkedsodium carboxymethyl cellulose) surface-active or dispersing agent.Moulded tablets may be made by moulding in a suitable machine a mixtureof the powdered compound moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach.

Compositions suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured base, usuallysucrose and acacia or tragacanth gum; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia gum; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Compositions for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter, gelatin,polyethylene glycol.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bactericides and solutes which render thecomposition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose scaled containers, for example, ampoules andvials, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Preferred unit dosage compositions are those containing a daily dose orunit, daily sub-dose, as herein above described, or an appropriatefraction thereof, of the active ingredient.

It should be understood that in addition to the active ingredientsparticularly mentioned above, the compositions of this invention mayinclude other agents conventional in the art having regard to the typeof composition in question, for example, those suitable for oraladministration may include such further agents as binders, sweeteners,thickeners, flavouring agents disintegrating agents, coating agents,preservatives, lubricants and/or time delay agents. Suitable sweetenersinclude sucrose, lactose, glucose, aspartame or saccharine. Suitabledisintegrating agents include corn starch, methylcellulose,polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.Suitable flavouring agents include peppermint oil, oil of wintergreen,cherry, orange or raspberry flavouring. Suitable coating agents includepolymers or copolymers of acrylic acid and/or methacrylic acid and/ortheir esters, waxes, fatty alcohols, zein, shellac or gluten. Suitablepreservatives include sodium benzoate, vitamin E, alpha-tocopherol,ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.Suitable lubricants may include magnesium stearate, stearic acid, sodiumoleate, sodium chloride or talc. Suitable time delay agents may includeglyceryl monostearate or glyceryl distearate.

One or more embodiments of the present invention may also providemethods, compositions agents or compounds which have an advantage over(or avoid a disadvantage) associated with known methods, compositions,agents or compounds used in the chemotherapeutic treatment of cancerousconditions or conditions associated with the hyperproliferation ofcells. Such advantages may include one or more of: increased therapeuticactivity, reduced side effects, reduced cytotoxicity to non-cancerous ornon-proliferative cells, improved solubility or dispersibilty forformulation into pharmaceutical compositions, improved stability or amore readily available means of obtaining said compounds, eg. bysimpler, cheaper or higher yielding synthetic or isolation processes.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications which fall within thespirit and scope. The invention also includes all of the steps,features, compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations of any two or more of said steps or features.

The references and citations disclosed within this specification aretaken to be incorporated herein in their entirety.

The invention will now be described with reference to the followingExamples which are included for the purpose of illustrating embodimentsof the invention and not to be construed as limiting the generalityhereinbefore described.

EXAMPLES Example 1

Isolation of Compounds A and B from Aglaia Leptantha

Compounds A and B were isolated using the following procedure:

(a) Treat a sample of ground bark from the tree species Aglaia leptanthawith methanol.

(b) Filter the extract and concentrate the resulting solution undervacuum.

(c) Fractionate the extract via solid-phase extraction on a C-18 Varianextraction column (10 g) using 0.1% formic acid in acetonitrile/waterwith increasing acetonitrile concentrations.

(d) Collect the eluate obtained with an acetonitrile/water ratio of7:20. Compounds A and B have a UV absorption maximum of 200, 273 run(acetonitrile/water/0.1% formic acid) and a HPLC retention times ofapproximately 30.67 (Compound A) and 31.05 minutes (Compound B) underthe following conditions:

C-8 Symmetry column (WATERS), 4.6×250 mm, 5 μm, 1 mL/min, lineargradient from 0% to 90% acetonitrile in water in 40 minutes with 0.1%formic acid.

(e) Concentrate fraction obtained under step (d).

(f) Chromatograph the concentrate obtained under step (e) on a C-18preparative column (WATERS, Nova-Pak C-18, 6 micron, 2.5×25 cm) at aflow rate of 20 mL/min using a linear gradient from 25% to 45%acetonitrile in water in 30 minutes with 0.1%

(g) Collect and concentrate the eluates with the chromatographic andspectroscopic characteristics outlined in step (d) at approximately 22minutes.

(h) Chromatograph each eluate obtained under (g) on a Sephadex LH20column using methanol as a solvent. Collect and concentrate thefractions with spectral characteristics outlined in (d). These sampleswere used for the structural elucidation of Compounds A and B.

(i) Alternatively to steps (b), (c) and (d), the methanol extractobtained under (a) may be partitioned with equal volumes of water anddichloromethane. The dichloromethane phase is then processed accordingto steps (e) to (h).

The compounds thus obtained have the following spectroscopic andphysical characteristics;

UV/V is absorption maxima: 223, 275 nm (in MeCN/H₂O, 0.1% HCOOH).

MS; Mass spectra were obtained on a Finnigan LCQ iontrap massspectrometer using the ESI source in the positive ion mode. The samplewas dissolved in 0.1%FA in MeOH and introduced into the source byinfusion with a syringe pump at rate of 3 μL/min. For Compounds A,signals were observed at m/z 677 [M+Na]⁺; MS² yielded m/z 659[M+Na-H₂O]⁺; MS³ yielded m/z 627 (loss of 32 amu); MS⁴ yielded m/z 595(loss of another 32 amu) and m/z 451 (loss of 176 amu, equivalent to thedioxane sidechain). For compound B signals were observed in the positiveion mode at m/z 677.2 [M+Na]⁴; MS² yielded product ions at m/z 627.2 andm/z 659.2. Further fragmentation of the signal at m/z 627.2 yielded aproduct ion at m/z 595.3.

Accurate mass spectra for Compound A were obtained on the Bruker 47cFourier Transform-Ion Cyclotron Resonance Mass Spectrometer (FTMS)fitted with an Analytica Electrospray Source (ESI). The sample wasdissolved in MeOH and introduced in to the source by direct infusionwith a syringe pump at a rate of 60 μL/min. The source was operated withcapillary voltage of 100 v. One signal was observed at m/z 677.2194[M+Na]⁺ meas.; C₃₄H₃₈O₁₃Na⁺ requires 677.2204.

NMR

The NMR spectra of Compounds A and B (see Formula (ii) below) wereacquired on 400 and 500 MHz Varian INOVA NMR spectrometers, in CD₃OD andCDCl₃, respectively. The following experiments were conducted: ¹H, ¹³C,DEPT, HMQC, HMBC, COSY. The ¹H NMR chemical shifts (obtained in CDCl₃)and the ¹³C NMR chemical shifts are listed in Table 1.

TABLE 1 (i)

Compounds A and B ¹H and ¹³CNMR shifts for Compounds A and B(Preliminary Position Assignments) Compound A Compound B Position 1H NMR¹³ C NMR ¹ H NMR ¹³C NMR Assignments (ppm) (ppm) (ppm) (ppm) 1 CH 5.03,d, 6.7 Hz, 1H 79.6 5.04, d, 6.8 Hz, 1H 79.8 2 CH 3.89. dd, 14.2, 6.7 Hz,1H 50.03 3.9, dd. 14. 6.8 Hz. 1H 50 COOCH₃ 170.6 170.7 COOCH₃ 3.65, s,3H 52.06 3.66, s, 3H 52 3 CH 4.28, d, 14.2 Hz, 1H 55.03 4.28, d, 14 Hz,1H 55 3a C 101.9 101.8 4a C 160.6 160.2 5 CH 6.43. d, 2 Hz, 1H 92.8 GAS,d. 2 Hz. 1H 92.8 6 C 160 159.8 OCH₃ 3.87. s, 3H 55.9 3.86, s, 3H 55.8 7CH 6.28 d, 2 Hz, 1H 93.9 6.29 d, 2 Hz, 1H 94.3 8 C 157.1 157.1 8a C109.6 109.4 8b C 93.4 1′ C 126.2 126.2 2′, 6′ 2xCH 7.10, brd, 9 Hz, 2H128.9 7.10, brd, 9 Hz, 2H 128.9 3′, 5′ 2xCH 6.68, brd, 9 Hz, 2H 112.76.69, brd, 9 Hz, 2H 112.8 4″ C 158.8 158.8 OCH₃ 3.71, s, 3H 55.05 3.72,s, 3H 55 1″ C 136.7 136.6 2″, 6″ 2xCH 6.84, m, 2H 127.8 6.86, m, 2H127.5 3″, 5″ 2xCH 7.06, m, 2H 127.8 7.06, m, 2H 127.5 4″ CH 7.06. m, 1H126.6 7.06, m, 1H 126.6 1′′′ CH 5.28, s, 1H 94 5.26, s, 1H 93.4 2′′′ CH4.59, s, 1H 95.2 4.60, s, 1H 95.2 OCH₃ 3.49, s, 3H 55.1 3.5, s, 3H 553′′′ CH₂ 4.13, t, 11.2 Hz, 1H 59 4.02, t, 11.2 Hz, 1H 59.6 3.56, dd,11.7,2 Hz, 1H 3.78. dd, 11.7, 2.4 Hz, 1H 4′′′ CH 4.23, brt, 11.3 Hz, 1H68.3 4.12, ddd, 11, 6.8, 2.8 Hz, 67.6 1H 5′′′ CH 3.61, m. 1H 70.6 3.66,m, 1H 71.4 6′′′ CH₂ 3.61, m, 2H 63.3 3.61, dd, 10.4, 4.4 Hz, 1H 62.53.72, m, 1H

Example 2

Determination of the substitution position of the dioxanyl sidechain onthe cyclopentabenzofurane core of Compounds A and B (Acetylation ofCompounds A and B)

The objective of this experiment was to unambiguously determine theattachment position of the dioxanyl sidechain to thecyclopentabenzofuran core in Compounds A and B.

Compounds A and B were dissolved in anhydrous pyridine (A: 4.2 mg in 280μL; B: 3 mg in 400 μL) and acetic anhydride was added (A: 140 μL, B: 200μL). The reaction mixtures were stirred under an argon atmosphere for 14(A) and 22 (B) hrs, respectively. The solvents were removed underreduced pressure to afford the diacetates as an orange residue (A 5.8mg; B: 3 mg). Purification of the crude residues was achieved by silicagel column chromatography eluting with 60% ethylacetate/petrol. Thediacetate of Compound A, Compound A′ (Formula (iii)), was obtained in68% yield (3.2 mg), and the diacetate of Compound B, Compound B′(Formula (iii)), was obtained in 41% yield (1.4 mg).

The purity of the two reaction products was assessed by reversed phaseHPLC using the same instrumentation as outlined in Example 1 (column:Xterra C-18, 1 mL/min, gradient: from 0 to 100% MeCN in 40 mins). Thestructures of compounds A′ and B′ were elucidated by electrospray MS and1D and 2D NMR experiments using the same conditions as described inExample 1. NMR spectra of Compounds A′ and B′ were obtained in CDCl₃with 500 and 400 MHz Varian INOVA instruments.

Both compounds yielded a single peak in the HPLC analysis with retentiontimes of 26.3 mins for Compound A′, and 27.7 mins for Compound B′.Compounds A′ and B′ showed positive molecular ions at m/z 761 [M+Na]⁺and 1499 [2M+Na]⁺ indicative of a molecular formula C₃₈H₄₂O₁₅. 1 and 2 DNMR experiments (¹H, HMQC and HMBC, NOESY) revealed that the twohydroxyl functions on the dioxanyl sidechain were acetylated. The ¹H and¹³C NMR chemical shifts are summarized in Table 2, and the NOESY spectrain Table 3.

The HMBC experiments of both diacetates show clear correlations of theproton signals of H-5, H-7 and H-1′″ to the carbon 6 of the aromaticring. The proton signals of H-7 and a methoxy group are correlated tothe carbon 8. This clearly indicates that the dioxanyl sidechain isattached at the position C-6 of the cyclopentabenzofuran core.

Further support for the position of the dioxanyl sidechain was derivedfrom the NOESY spectra of both compounds. The NOE signals are observedfrom both H-5 and H-7 to H-1′″ of the dioxanyl side chain, and only H-7shows a NOE signal to the C-8 methoxy signal. The NOE signals observedin the dioxanyl ring systems of the two compounds clearly indicate thatthey differ in regard to the stereochemistry of the di-hydroxyethanesidechain. The NOE signals for the cyclopentabenzofuran core are inagreement with published data and confirm the stereochemistry depictedin Tables 1 and 2.

TABLE 2 (iii)

Compounds A′ and B′ (Diacetates of Compounds A and B) ¹H and ¹³C NMRchemical shifts for Compounds A′ and B′ Compound A′ Compound B′(Diacetate of Compound A) (Diacetate of Compound B) Position Assignment¹HNMR ¹³C NMR ¹H NMR ¹³C NMR 1 CH 5.06, d, 8 Hz 19.8 5.07, d, 8 Hz 79.82 CH 3.89, under OCH₃-8 50.4 3.87, under OCH₃-8 50 2 COOCH₃ 170.4 170.42 COOCH₃ 3.64, s 51.9 3.63, s 51.8 3 CH 4.26. d. 14 Hz 55 4.27. d, 14.4Hz 54.8 3a C 101.9 102 4a C 160.6 160.5 5 CH 5.43, d, 2 Hz 93.3 6.45, d,2 Hz 93.2 6 C 159.6 159.7 7 CH 6.27 d, 2 Hz 93.2 6.29, d, 2 Hz 93 8 C157 156.9 S OCH₃ 3.89. s 56 3.88, s 55.8 Ba C 109.8 109.6 Sb C 93.5 93.4Sb OH 2.35,s 223 1′ C 126.2 126.4 2′, 6′ 2xCH 7.10, brd, 9 Hz 129.17.10, brd, 9 Hz 129 3′, 5′ 2xCH 6.67, brd, 9 Hz 112.7 6.68, brd, 9 Hz112.6 4′ C 158.8 158.6 4′ OCH₃ 372, s 55 3.72, s 55 1″ C 136.8 136.6 2″,6″ 2xCM 6.83, m 127.7 6.86, m 127.8 3″, 5″ 2xCH 7.05,m 127.1 7.05, m127.8 4″ CH 7.05, m 126.6 7.05, m 126.6 1′′′ CH 5.38, s 93.7 5.31, s93.2 2′′′ CH 4.61, s 95.2 4.62, s 95.2 2′′′ OCH₃ 3.50, s 55.2 3.5, s, 3H55 3′′′ CH₂ 3.94, t, 11.2 Hz 58.8 3.93, t, 12 Hz 59.5 3.54, dd, 11.2, 3Hz 3.59, dd, 12, 2.5 Hz, 4′′′ CH 4.38, dt, 11, 3 Hz 66.3 4.37, td, 9,2.5 Hz 64.9 5′′′ CH 5.12, td, 6, 3 Hz 69.1 5.00, ddd, 9, 4, 2.5 Hz 70.26′′′ CH₂ 4.22, dd, 11.2, 6 Hz 61.3 4.21, dd, 12.4, 4 Hz, 61.5 3.88, ?under 4.12, dd, 12.4, 2.5 Hz OCH₃-8 5′′′ COCH₃ 170.3 169.9 5′′′ COCH₃2.14, s 20.8 2.07 20.8 6′′′ COCH₃ 170.7 170.8 6′′′ COCH₃ 1.79 20.4 1.7420.1

TABLE 3

Comparison of NOESY Spectra of Compounds A′ and B′ Compound A′ CompoundB′ 1H NMR NOEs 1H NMR NOEs 6.43, d, H-5 5.38, s, H-1′′′ 6.45, d, H-55.31, s, H-1′′′ 4.38, dt, H-4′′′ 4.37, td, H-4′′′ 4.21, dd, H-6′′′-11.74, s, COCH₃-6′′′ 6.27, d, H-7 5.38, s, H-1′′′ 6.29, d, H-7 5.31, s,H-1′′′ 4.21, dd, H-6′′′-1 3.89, s, OCH₃-8 5.38, s, H-1′′′ 4.38, dt,H-4′′′ 5.31, s, H-1′′′ 4.37, td, H-4′′′ 3.89, s, OCH₃-8 3.88, s, OCH₃-83.50, s, OCH₃-2′′′ 3.5, s, OCH₃-2′′′

Example 3

Compounds A and B are cytostatic and cytotoxic for human tumour celllines

(a) Compounds A and B were identified from a bark sample of Aglaialeptantha through their ability to inhibit production of Tumour NecrosisFactor-α (TNF-α) by THP-1 human promonocytic leukemia cells (Tsuchiya,et al, Int. J. Cancer, 1980, 26(2):171-6) activated withlipopolysaccharide (LPS). Table 4 summarises the results comparing theactivity of Compounds A and B for inhibition of TNF-α production totheir effects on general cell metabolism measured using WST-1 reduction,DNA synthesis and protein synthesis assays for THP-1 cells. Compounds Aand B potently inhibited TNF-α production at broadly similarconcentrations that were active in the WST-1 reduction, DNA and proteinsynthesis assays. For comparison, the effects of Compounds A and B onA549 lung epithelial carcinoma cells (Leiber et al, Int. J. Cancer,1976, 17(1)-62-70) were also measured and the data is also included inTable 4. Compounds A and B are significantly less potent for inhibitionof interleukin-1 (IL-1)-induced Intercellular Adhesion Molecule-1(ICAM-1) expression by A549 cells even though in these cells the proteinand DNA synthesis inhibition occur at broadly similar concentrations asfor THP-1 cells.

TABLE 4 Comparison of the effects of Compounds A and B in THP-1 and A549Cells* IC₅₀ (μM) THP-1 cells A549 cells TNF-a WST-1 Protein DNA ICAM-1Protein DNA Compound Production Reduction SynThesis Synthesis ProductionSynthesis Synthesis Compound A 0.06 0.03 0.06 0.015 2 0.02 0.007Compound B 0.015 0.04 0.003 0.003 5 0.01 0.004 *Purified Compound A orCompound B solubilized in DMSO were tested over a range ofconcentrations in parallel for inhibitory activity in the various assaysin both THP-1 and A549 cells. The concentration that resulted in a 50%inhibition of the relevant response (IC₅₀) is shown. Production of TNFαby THP-1 cells was measured as that released into the culturesupernatant over 18 hours by sandwich enzyme-linked immunosorbent assay# (ELISA) using the following mouse anti-TNFα monoclonal antibodies(capture antibody, MAB610; detection antibody, biotinylated MAB210; bothfrom R&D Systems, Minneapolis MN, USA). Surface expression of ICAM-1 byA549 cells was assayed after 24 hours of culture by direct antibodybinding using a europium-labelled mouse anti-ICAM-1 monoclonal antibody(R&D Systems Cat No. BBA3) and measured by time-resolved fluorescenceusing Delfia assay # (EG&G Wallac, Turku, Finland). Reduction of WST-1(Roche, Cat. No. 1644807) by THP-1 cells was measured after 18 hours ofculture according to the manufacturer's instructions. Protein synthesiswas measured as the uptake of [¹⁴C]-leucine (0.5 μCi/mL) after 48 hoursfor THP-1 cells and 72 hours for A549 cells cultured in growth medium(RPMI-1640, 10% FBS) containing 10% the usual L-leucine concentration (5mg/mL). DNA synthesis # was measured as the uptake off [¹⁴C]-thymidine(0.5 μCi/mL) after 48 hours for THP-1 cells and 72 hours for A549 cellsin normal growth medium.

(b) Compound A was assessed for cytotoxic and cytostatic activityagainst a panel of cell lines derived from a variety of human tumourtypes in addition to THP-1 and A549 cells (Table 5). These included K562leukemic cells (Lozzio and Lozzio, 1975, Blood 45:321-34), PC3 prostatetumour cells (Kaighn et al., 1979, Invest. Urol. 17:16-23) and SF268glioblastoma cells (Westphal et al, 1985, Biochem. Biophys. Res.Commun., 132:284-9). Compound A exhibited potent cytostatic activity innearly all cell lines tested with GI₅₀ values ranging between 1-7 nM.Compound A also exhibited potent cytotoxic effects against the varioustumour cell lines. Interestingly, the THP-1 and PC3 cells proved themost rapidly killed with little difference in LC₅₀ values obtained after3 or 6 days of culture. However, the cytotoxic potency of Compound Aincreased dramatically after 6 days of culture for the K562, A549 andSF268 cells. It should be noted that the concentration of Compound Arequired to inhibit cell proliferation were significantly lower thanthose required to elicit a cytotoxic response. Hence, the cytostaticeffect of Compound A is biochemically distinguishable from its abilityto induce cell death. Table 6 shows that Compound B exhibited cytotoxiceffects against the various tumour cell lines with comparable potency tothat observed with Compound A.

TABLE 5 Compound A has potent cytostatic and cytotoxic activity invarious human tumour cell lines in vitro* Compound A GI₅₀ (nM) LC₅₀ (nM)LC₅₀ (nM) Tumour Tumour (3 day (3 day (6 day Source Cell Line cultures)cultures) cultures) Leukemia THP-1 — 36 24 K562 1 >1000 10 Lung A549 7914 21 Prostate PC3 5 18 12 Brain SF268 3 461 29 *Purified Compound Awas tested over a range of concentrations up to a maximum of 1 × 10⁻⁶ M(1000 nM) for cytostatic and cytotoxic activity against a panel of celllines derived from various human tumour types as indicated. The GI₅₀value represents the concentration of compound that inhibited the cellnumber increase (relative to untreated cells) by 50% after 3 # days ofculture. Relative cell number was determined by measuring cellular DNAusing a fluorescent DNA-binding dye (YOYO-1) after lysing the cells withdigitonin (Becker et al., Anal Biochem, 1994, 221(1):78-84). The LC₅₀value represents the concentration of compound that killed 50% of thecells. Cell death was measured as the proportion of dead # cellsexhibiting sub-diploid DNA content determined by flow cytometry afterstaining with propidium iodide (Nicoletti et al., J. Immunol. Methods,1991, 139:271-79).

TABLE 6 Compounds A and B exhibit similar cytotoxic activity* LC₅₀ (nM)(6 day cultures) Tumour Tumour Compound Compound Source Cell Line A BLeukemia THP-1 11 15 K562 12 15 Lung A549 15 12 Prostate PC3 12 12 BrainSF268 12 22 *The cytotoxic activity of Compounds A and B were comparedfor the various tumour cell lines as described in Table 5.

(c) Testing of Compound A against a much larger cell line panel in theNCI in vitro anticancer screen (Table 7) confirmed the results describedabove. Using a different assay methodology based on measurement of totalcellular protein the results confirm that Compound A had broad andpotent cytostatic effects with all of the cell lines exhibiting maximalinhibition of cell growth even at the lowest dose tested (10 nM).Consistent with the data in Table 5 the cytotoxic effects measured after2 days of culture were more varied with LC₅₀ values ranging from 10 nMfor COLO-205 colon tumour cells to ˜90 μM for 786-0 renal tumour cells.These data indicate that Compound A had potent in vitro activity againsta wide range of tumour cell lines representing a variety of differentmajor types of cancer including leukemia, lung, colon, brain, melanoma,ovarian renal, prostate and breast tumours.

TABLE 7 Activity of Compound A measured in the NCI in vitro anticancerdrug discovery screen* Tumour Source Tumour Cell Line GI₅₀ (nM) LC₅₀(nM) Lung EKVX <10  23 Lung NCI-H226 <10 193 Lung NCI-H460 <10 38,019Lung NCI-11522 <10  2,399 Colon COLO-205 <10  10 Colon HT29 <10  1,000Brain SF-268 <10  1,000 Brain SF-295 <10  1,230 Brain SF-539 <10  1,096Brain SNB-75 <10  54 Melanoma LOX IMV1 <10  1,000 Melanoma MALME-3M <1023,988 Melanoma M14 <10  51 Melanoma SK-MEL-2 <10 19,055 MelanomaSK-MEL-28 <10  2,661 Melanoma 8K-MEL-5 <10  67 Melanoma UACC-62 <10  30Ovarian IGROVI <10  2,600 Ovarian OVCAR-4 <10  11 Ovarian OVCAR-5 <10 1,000 Ovarian OVCAR-8 <10 21,878 Ovarian SK-OV-3 <10 82,224 Renal 786-0<10 91,201 Renal A498 <10 18,621 Renal ACHN <10 31,623 Renal RXF 393 <10 1,641 Breast MCF7 <10  1,000 Breast MDA-MB-231/ATCC <10 25,410 BreastMDA-MB-435 <10  45 Breast MDA-N <10 543 Breast BT-549 <10 32,734*Compound A was tested for activity in the National Cancer Institute invitro anticancer drug discovery screen. For this Compound A was testedat five 10-fold dilutions ranging from 10⁻⁴M to 10⁻⁸M against a panel ofdifferent human tumour cell lines representing major types of cancer asdescribed by Boyd and Paull, Drug Development Research, 1995, 34:91-109.Briefly, this involved # a 48 hr incubation of the cells with Compound Aprior to measuring the relative cell number by staining withsulforhodamine B. GI₅₀ values represent the concentration of Compound Athat inhibited net growth of the cells by 50% compared to untreatedcontrols. LC₅₀ values represent the concentration of Compound A thatresulted in a # net 50% loss (killing) of the cells relative to thestart of the experiment. The data represent the average values from twosuch experiments conducted.

Example 4

Cytotoxic activity of Compound A is not shared by other known relatedcompounds lacking dioxanyloxy substitution. Compound A′ displayscytotoxic activity.

(a) Table 8 compares the cytostatic and cytotoxic effects of Compound Ato three previously identified 1H-cyclopenta[b]benzofuran lignans thatlack the dioxanyloxy group at the C6-position. The reference compoundsare: Rocaglaol (Reference Compound 1) (Ohse et al., J Nat Prod, 1996,59(7):650-52); 4′-Demethoxy-3′,4′-methylenedioxyrocaglaol (ReferenceCompound 2) and Methyl 4′-demethoxy-3′,4′-methylenedioxyrocaglate(Reference Compound 3) (Lee et al., Chem Biol Interact, 1998,115(3):215-28). All four compounds exhibited detectable cytostaticactivity in A549 cells with Compound A being the most potent followed indecreasing order by Reference Compounds 3, 2 and 1 respectively.Importantly, of the compounds tested, other than Compound A none of theReference Compounds exhibited any appreciable cytotoxicity in eitherTHP-1 or A549 cells at doses up to 5000 nM over the 3 day assay. Withoutintending to limit the invention by theory, it is suggested that thenovel dioxanyloxy substitution at the C6-position is important for thecytotoxic activity exhibited by Compound A and distinguishes it from anyother previously identified 1H-cyclopenta[b]benzofuran lignans.

TABLE 8

Related 1H-cyclopenta[b]benzofuran lignans lacking the novel dioxanyloxyside chain do not exhibit cytotoxic activity* A549 cells THP-1 cellsCompound GI₅₀ (nM) LC₅₀ (nM) LC₅₀ (nM) Compound A 13 514 15 ReferenceCompound 1 3980 >5000 >5000 Reference Compound 2 389 >5000 >5000Reference Compound 3 56 >5000 >5000 *A549 and THP-1 cells were treatedwith increasing concentrations of the various compounds up to a maximumof 5 × 10⁻⁶ M (5000 nM) and the effects on cell proliferation and cellviability were determined after 3 days of culture. GI₅₀ values weredetermined by measuring relative changes in cell number using YOYO-1 asdescribed for Table 5. LC₅₀ values were determined by measuring celldeath as a function of loss of memebrane integrity using YOYO-1 # uptake(Becker et al., Anal Biochem, 1994, 221(1): 78-84). The structures ofthe reference compounds are also shown.

(b) Table 9 shows that acetylation of the dioxanyl side chain ofCompounds A and B did not reduce their biological activity sinceCompounds A′ and B′ inhibited WST-1 reduction of THP-1 leukemic with atleast similar potencies to the unmodified compounds. The lower IC₅₀values for all the compounds depicted in this WST-1 reduction experimentcompared to the values shown in Table 4 reflects the enhancedsensitivity of the cells when treated for 3 days compared to the 18 hrtreatment used in the latter assay.

TABLE 9 Acetylation of the dioxanyl side chain of Compounds A and B doesnot inhibit their biological activity* Compound IC₅₀(nM) Compound A 2.0Compound A′ 0.3 Compound B 1.5 Compound B′ 0.7 *Purified compoundssolubilized in DMSO were tested over a range of concenfrations for theireffects on the reduction of WST-1 by THP-1 leukemic cells as describedfor Table 4 except that the cells were cultured in the presence of thevarious compounds for 3 days prior to measuring WST-1 reduction.

Example 5

Compound A has acute protein synthesis inhibitory activity

Compound A was also examined to determine whether it could rapidlyinhibit general protein biosynthesis. Using [¹⁴C] leucine incorporationinto insoluble cellular material as an assay for general proteinbiosynthesis, Table 9 shows that Compound A had an inhibitory effectevident within 3 hrs after addition to THP-1 cells with an IC₅₀ of ˜30nM. DNA synthesis measured over the same time was also inhibited, butless potently (IC₅₀˜70 nM) and may be secondary to protein synthesisinhibition. Cyloheximide, a known protein synthesis inhibitor (Obrig etal, 1971, J. Biol. Chem. 246(1): 174-181), also inhibited both proteinand DNA synthesis with Compound A being significantly more potent thancycloheximide in its effects. Table 10 shows that Compound A alsoinhibited general protein synthesis in A549 cells with an IC₅₀ of ˜30 nMwhich is similar to that observed in the THP-1 cells

TABLE 10 Compound A inhibits general protein biosynthesis* IC₅₀ (nM)THP-1 cells A549 cells Protein DNA Protein Compound synthesis Synthesissynthesis Compound A 27 72 32 Cycloheximide 263 303 238 *THP-1 cells andA549 cells were pretreated with the indicated concentrations of CompoundA for 1 hour prior to the addition of (1 μCi/mL) [¹⁴C] leucine (proteinsynthesis) or [¹⁴C] thymidine (DNA synthesis) for a further 2 hours. TheIC₅₀ values represent the # concentration of Compound A required toinhibit incorporation of isotope by 50% relative to untreated controlcell cultures.

Example 6

Compound A induces differentiation of human leukemic cell lines.

In the experiments with the THP-1 monocytic leukemia cells, whichnormally grow unattached in suspension, we noticed that prolongedexposure of the cells to 10 nM Compound A resulted in accumulation ofcells that adhered to the plastic and exhibited numerous pseudopodia(FIG. 1). This is a morphology highly characteristic of maturemacrophages and similar morphological effects were observed when thecells were treated with other known inducers of macrophagedifferentiation including interferon-γ (IFNγ) or phorbol 12-myristate13-acetate (PMA). To investigate this further the effects of Compound Aon HL60 human promyelocytic leukemic cells (Collins, et al, Nature,1977, 270:347-9) were examined (Table 11). This widely used cell line iswell characterised as a model of human myelomonocytic differentiation(Collins, Blood, 1987, 70(5):1233-44). In this experiment monocyticdifferentiation was quantitated by measuring CD14 surface antigenexpression by flow cytometric analysis CD14, all LPS-binding protein, isexpressed on the surface of cells of the myelomonocytic lineage and isnormally expressed at very low levels in undifferentiated HL60 cells(Ferrero et al., Blood, 1983, 61(1):171-9). Consistent with the THP-1data above, Table 10 shows that Compound A at doses greater than 10 nMsignificantly enhanced CD14 expression in the viable HL60 cellsremaining after 4 days of culture. Taken together these data stronglyindicate that Compound A has the ability to induce differentiation ofhuman leukemic cell lines.

TABLE 11 Compound A promotes monocytic differentiation of HL60 leukemiccells* Compound A % cells expressing concentration (nM) CD14 0 1.3% 53.3% 10 5.7% 25 46.0% 50 43.0% *HL60 cells were cultured for 4 days withthe indicated concentration of Compound A then collected and fixed in70% ethanol. Cells were then stained with mouse monoclonal anti CD14antibody (OKM1) and this was measured using FITC-conjugated goatanti-mouse IgG1 as a secondary antibody. Stained cells were visualisedby flow cytometry and analysis # was restricted to cells judged viableat the time of fixing based on their forward and side light-scattercharacteristics. Non specific staining of cells was controlled for byincubating with secondary antibody only.

Example 7

Cytostatic activity of Compound A is associated with a generalinhibition of cell cycle progression in A549 cells

DNA content analysis of THP-1 cells treated with varying concentrationsof Compound A (FIG. 2) demonstrated that at 10 nM it was only weaklycytotoxic (increased accumulation of dead cells from 7% to 17%) andunder these conditions caused cells to accumulate in the G0/G1 phases ofthe cell cycle. This indicates that Compound A also has cytostaticactivity in THP-1 cells. For comparison, FIG. 2 shows that themicrotubule destabilising drug paclitaxel (Sorger et al., Curr Opin CellBiol, 1997, 9(6):807-14) which also induced THP-1 cell death, causedcells to accumulate in the G2/M phases of the cell cycle.

The cytostatic effect of Compound A on the proliferation of A549 cellswas confirmed by directly counting the number of cells at intervals overa nine day period (FIG. 3). When compared to untreated cells 10 nMCompound A prevented the increase in cell number by more than 95% withfewer than 10% dead cells observed at this time (measured by trypan blueexclusion). Thus, under these conditions the decreased cell number cannot simply be accounted for by increased cell death. A significantinhibition of cell number was seen within 2 days indicating thatCompound A acts in a rapid manner. At the higher concentrations of 50 nMand 250 nM Compound A had cytotoxic effects and increased cell death to86% and 100% respectively after 9 days and accounts for the decline incell number to levels below the original staring number at this time. Atthe non-cytotoxic concentration of 10 nM, Compound A has a rapid andpotent cytostatic effect on A549 cells.

To help identify a potential mechanism for the effects of Compound A,DNA content analysis was performed to determine where in the cell cycleit exerted its effect (FIG. 4). Cell cycle analysis of A549 cellstreated with Compound A for 6 days showed that at 10 nM, where noobvious cytotoxicity was evident, there was a minor decline in theproportion of cells in the G0/G1 phases of the cell cycle with aconcomitant increase in cells in the G2/M phases. Taken together withthe growth curve data in FIG. 3 above, these data indicate that 10 nMCompound A results in a general lengthening of all phases of the cellcycle with perhaps a slightly more pronounced elongation of the G2/Mphases. This contrasts to the effects of paclitaxel a drug known to actselectively at the G2/M phases of the cell cycle (FIG. 4). As theconcentration of Compound A was increased and its cytotoxic effectsbecame evident the proportion of cells in the S and G2/M phasesdecreased with a corresponding rise in cells in G0/G1 phases. Althoughthere was little difference in the number of dead cells between 50 nMand 250 nM the higher dose resulted in a greater accumulation of cellsin the G0/G1 phases of the cell cycle. Thus, compared to THP-1 cells(see FIG. 2) higher concentrations of Compound A are required to inhibitprogression through the G0/G1 phases of the cell cycle in A549 cells.

K562 leukemic cells treated with 10-15 nM Compounds A or B exhibited acharacteristic accumulation of cells in G2/M phases of the cell cycle(FIG. 5). This occurred over a narrow range of concentrations sinceCompounds A or B at less than 5-8 nM or more than 25 nM did not cause aG2/M phase accumulation. These data indicate that different cell linescan vary in their sensitivity and responses to Compounds A and B forcell cycle phase-specific effects.

Example 8

The cytostatic effect of Compound A is reversible in A549 cells

The reversibility of the effects of Compound A was determined. For this,A549 cells remained untreated or were cultured in the presence ofvarious concentrations of Compound A or with paclitaxel for 5 days priorto removal of the compounds and the cells cultured for a further 4 daysprior to determining cell number (FIG. 6). 10 nM of Compound Asignificantly suppressed the increased cell number for up to 9 dayswithout significant cytotoxicity. However, for these cultures whenCompound A was removed after 5 days there was over a five-fold increasein cell number over the subsequent 4 days of culture, representing 2-3population doublings. The effects of treatments which were deleteriousto the cells, such as higher concentrations of Compound A or thepresence of paclitaxel, were not reversed upon their removal.

Example 9

Compound A inhibits cell cycle-dependent cytotoxicity elicited byvarious anti-cancer agents

To further examine the cell cycle effects of Compound A a cytostaticconcentration of this compound was combined together with otheranti-cancer agents known to act at specific points in the cell cycle tosee if Compound A could perturb their cell cycle-dependent effects. Cellviability was assayed after 3 days by measuring exclusion of thefluorescent DNA-binding dye YOYO-1. (Becker et al., Anal Biochem, 1994,221(1):78-84). A549 cells were treated with 10 nM non-cytotoxic dose ofCompound A in the presence of increasing concentrations of camptothecinand paclitaxel. Camptothecin is an inhibitor of DNA topoisomerase 1, anenzyme required for DNA replication, and results in pertubation of the Sphase of the cell cycle with subsequent cell death due to activation ofan S phase checkpoint (Darzynkiewicz et al., Ann N Y Acad Sci, 1996,803:93-100). Paclitaxel, as already mentioned, inhibits microtubulefunction required for formation of the mitotic spindle thereby resultingin activation of an M phase checkpoint and subsequent cell death (Sorgeret al., Curr Opin Cell Biol, 1997 9(6):807-14). FIG. 7 shows that 10 nMCompound A significantly reduced the cytotoxic effects of bothcamptothecin and paclitaxel even when these drugs were added at up to a2000-fold excess. Compound A may, in a dominant manner, prevent the cellcycle-dependent cytotoxic effects of camptothecin and paclitaxel.

This was examined in more detail using DNA content analysis tospecifically measure cell cycle progression and cell death. In thisexperiment in addition to camptothecin and paclitaxel cells were alsotreated with vinblastin (another microtubule inhibitor) (Sorger et al.,1997, supra) and staurosporine (a kinase inhibitor) (Gescher, Crit RevOncol Hematol., 2000, 34(2):127-35). As previously found, A549 cellstreated with 10 nM Compound A showed a minor decrease in cells in G0/G1with a slight increase in G2/M phase cells with no detectable increasein cell death over the three days of culture (FIG. 8). Consistent withits known mechanism of action camptothecin resulted in accumulation ofcells in S phase of the cell cycle and also increased the level of deadcells detected as those with a sub-diploid DNA content. Also asexpected, both vinblastin and paclitaxel resulted in the majority ofcells arresting in the G2/M phases of the cell cycle and increasedappearance of sub-diploid dead cells. However, for all of these agentsthe presence of 10 nM Compound A prevented their characteristic cellcycle arrest and significantly inhibited their cytotoxic effects,dramatically reducing the appearance of sub-diploid dead cells. Incontrast, Compound A had little effect on the cytotoxic effects ofstaurosporine, an agent which appears capable of killing cells at allactive phases of the cell cycle.

Example 10

Cytostatic effects of Compound A do not correlate with a biomarker forreplicative senescence.

The dramatically decreased growth rate of A549 cells cultured in thepresence of 10 nM Compound A (see FIG. 3) led to the consideration ofthe possibility that this compound was inducing replicative senescenceof these immortal tumour cells. Consistent with this possibility underthese conditions A549 cells with a morphology highly suggestive of asenescent phenotype were often observed, being highly flattened with anenlarged surface area compared to their usual appearance (compare forexample FIG. 9 subpanels a and b). This was evaluated further bymeasuring senescence-associated β-galactosidase (SA-β-gal) activity, abiomarker previously described to correlate well with senescence ofhuman cells (Dimri et al., Proc Natl Acad Sci USA 1995 92(20):9363-7).Recently, it has been found that some anti-cancer agents that act bydiverse mechanisms, including doxorubicin, cisplatin, cytarabine,etoposide and paclitaxel, can all induce SA-β-gal activity in a varietyof tumour cell lines (Chang et al., Cancer Res 1999, 59(15):3761-7).Therefore, in addition to Compound A A549 cells were also treated withdoxorubicin as an experimental control. This drug acts by stabilisingDNA/topoisomerase II complexes thereby causing DNA damage which resultsin subsequent S phase cell cycle arrest and/or cell death(Froelich-Ammon and Osheroff, 1995, J. Biol. Chem. 270(37):21429-21432).FIG. 9 shows that consistent with the earlier report A549 cells treatedwith 250 nM doxorubicin displayed the flattened enlarged phenotype ofsenescent cells and exhibited SA-β-gal activity. In contrast, Compound Aat various doses from 10-50 nM failed to induce SA-β-gal activity eventhough the cells exhibited the flattened enlarged morphology. Thus, incontrast to a variety of other anti-cancer drugs the cytostatic effectsof Compound A do not correlate with this particular marker of cellsenescence.

Example 11

Compound A inhibits cell proliferation but not increased cell size

It is well known that cell proliferation and cell growth reflected asincreased mass of individual cells are biochemically separable processes(Pardee, Science, 1989, 246:603-8). Although at certain concentrationsCompound A can inhibit cell proliferation without overt cytotoxicity itwas also evaluated whether Compound A also affected cell growth. Forthese experiments A549 cells were treated with various non-cytotoxicdoses of Compound A up to 10 nM and the relative cell size determinedafter 6 days of culture by measuring forward light scatter using a flowcytometer. The data depicted in Table, 11 show that in the presence ofCompound A A549 cells exhibited an increase in the mean forward scatterby over 20%. This occurred only at concentrations which are cytostaticfor this cell type.

TABLE 12 Compound A increases cell size* Compound A % increase in meanconcentration (nM) cell volume 0 — 2.5 10.4% 5.0 10.7% 10.0 22.4% *A549cells cultured for six days with the various non-cytotoxicconcentrations of Compound A as indicated were examined by flowcytometry for their forward light scatter characteristics which directlyrelates to cell size. The % increase in mean cell volume represents therelative change in the mean forward scatter value for the treated versusuntreated cell populations.

Example 12

Compound A inhibits growth of human tumour cell lines in a mousexenograft tumour model.

The ability of Compound A to inhibit growth of human tumour cells invivo was assessed using male athymic mice injected subcutaneously in thedorsal flank region with 2×10⁶ PC3 human prostate tumour cells. CompoundA administration (3 mg/kg) by intraperitoneal injection commenced aftereight days once the PC3 tumour was palpable and continued three times aweek until 29 days after the initial inoculation of the tumour cells. Atthis time all mice were killed and tumours excised and weighed. FIG. 10Ashows that compared to the control animals treated with vehicle alonethe mice treated with Compound A displayed a greatly reduced increase inmean tumour volume over the course of the experiment. This was confirmedat the end of the experiment when tumours were excised and weighed itwas found that Compound A treatment reduced the mean tumour weight by˜60% (FIG. 10B). Body weight was unaffected with both control andtreated groups exhibiting a similar ˜12% decrease in mean body weightover the duration of the experiment. Thus, Compound A exhibits in vivoantitumour activity.

We claim:
 1. A compound of Formula (I):

wherein: each R⁴-R¹⁰ is independently selected from the group consistingof hydrogen, optionally substituted alkyl, optionally substituted acyl,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted cycloalkylalkyl, optionally substituted arylacyl,optionally substituted cycloalkylacyl and a C-1 linked saccharide; X isOR⁸ or NR⁹R¹⁰; R¹¹ and R¹² are each independently hydrogen or, OR⁴ andR¹¹, and/or OR⁵ and R¹² together form a methylenedioxy group; and Y isselected from the group consisting of optionally substituted phenyl,optionally substituted benzyl, optionally substituted benzoyl,optionally substituted C₃-C₈ cycloalkyl, optionally substitutedCH₂-(C₃-C₈ cycloalkyl), optionally substituted 5-6 membered heterocyclyland optionally substituted CH₂-(5-6 membered heterocyclyl); or a salt,isomer or prodrug thereof.
 2. The compound according to claim 1 whereinR⁴ and Y are independently selected from the group consisting ofoptionally substituted C₃-C₈ cycloalkyl, optionally substituted benzoyl,optionally substituted benzyl, optionally substituted CH₂-(C₃-C₈cycloalkyl) and C-1 linked saccharide.
 3. The compound according toclaim 1 wherein the optionally substituted C₃-C₈ cycloalkyl group isoptionally substituted C₅-C₆-cycloalkyl and the optionally substitutedCH₂-(C₃-C₈ cycloalkyl) group is optionally substituted CH₂-(C₅-C₆cycloalkyl).
 4. The compound according to claim 1 wherein Y is anoptionally substituted 5-6 membered heterocyclyl group or an optionallysubstituted C₅-C₆ cycloalkyl group.
 5. The compound according to claim 4having Formula (i)

or isomer thereof, wherein: each R¹-R¹⁰ is independently selected fromthe group consisting of hydrogen, optionally substituted alkyl,optionally substituted acyl, optionally substituted aryl, optionallysubstituted arylalkyl, optionally substituted cycloalkylalkyl,optionally substituted arylacyl, optionally substituted cycloalkylacyland a C-1 linked saccharide; X is OR⁸ or NR⁹R¹⁰, and R¹¹ and R¹² areeach independently hydrogen or OR⁴ and R¹¹, and/or OR⁵ and R¹² togetherform a methylenedioxy group.
 6. The compound according to claim 1 or 5wherein R⁸ is selected from the group consisting of hydrogen, C₁₋₆alkyl, phenyl, benzyl and C-1 linked saccharide.
 7. The compoundaccording to claim 1 or 5 wherein R⁹ and R¹⁰ are independently selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, phenyl and benzyl. 8.The compound according to claim 1 or 5 wherein R¹¹ and R¹² are bothhydrogen.
 9. The compound according to claim 5 wherein each of R¹R⁷ areindependently selected from the group consisting of hydrogen, methyl,ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t-butyl,cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl,cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl,phenyl, benzyl, acetyl and C-1 linked saccharide.
 10. The compoundaccording to claim 5 wherein R¹ and R² are both hydrogen.
 11. Thecompound according to claim 5 wherein R³ is methyl.
 12. The compoundaccording to claim 5 wherein at least one of R³-R⁵ is methyl, ethyl orpropyl.
 13. The compound according to claim 12 wherein at least one ofR³-R⁵ is methyl.
 14. The compound according to claim 12 wherein at leasttwo of R³-R⁵ are methyl, ethyl or propyl.
 15. The compound according toclaim 14 wherein at least two of R³-R⁵ are methyl.
 16. The compoundaccording to claim 14 wherein all of R³-R⁵ are methyl, ethyl or propyl.17. The compound according to claim 16 wherein all of R³-R⁵ are methyl.18. The compound according to claim 1 or 5 wherein R⁶ and R⁷ are bothhydrogen.
 19. The compound according to claim 1 or 5 wherein X is OR⁸and where R⁸ is selected from the group consisting of hydrogen, methyl,ethyl and propyl.
 20. The compound according to claim 19 wherein R⁸ ismethyl.
 21. The compound according to claim 1 or 5 wherein X is NR⁹R¹⁰where R⁹ and R¹⁰ are both hydrogen or methyl; or R⁹ and R¹⁰ aredifferent but at least one of R⁹ or R¹⁰ is hydrogen and the other isC₁₋₆ alkyl.
 22. The compound according to claim 21 wherein C₁₋₆ alkyl ismethyl, ethyl or propyl.
 23. The compound according to claim 5 havingFormula (ii):

or isomer thereof.
 24. The isomer of the compound of claim 23 having thefollowing NMR spectral characteristics: ¹H NMR (CDCl₃, ppm) 3.49, s, 3H;3.56, dd, 11.7, 2 Hz, 1H; 3.61, m, 1H, 3.61, 2H; 3.65, s, 3H; 3.71, s,3H; 3.87, s, 3H; 3.89, dd, 14.2, 6.7 Hz, 1H; 4.13, t, 11.2 Hz, 1H 4.23,brt, 11.3 Hz, 1H; 4.28, d, 14.2 Hz, 1H; 4.59, s, 1H; 5.03, d, 6.7 Hz, 1Hz; 5.28, s, 1H; 6.28, d, 2 Hz, 1H; 6.43, d, 2 Hz, 1H; 6.68, brd, 9 Hz,2H; 6.84, m, 2H; 7.06, m, 2H, 7.06, m, 1H; 7.10, brd, 9 Hz, 2H; ¹³C NMR(CDCl₃), (ppm) 50.03, 52.06, 55.03, 55.05, 55.1, 55.9, 59, 63.3, 68.3,70.6, 79.6, 92.8, 93.4, 93.9, 94, 95.2, 101.9, 109.6, 112.7, 126.2,126.6, 127.8, 127.8, 128.9, 136.7, 157.1, 158.8, 160, 160.6, 170.6. 25.The isomer of the compound of claim 23 having the following NMR spectralcharacteristics: ¹H NMR (CDCl₃, ppm) 3.5, s, 3H; 3.61, dd, 10.4, 4.4 Hz,1H; 3.66, m, 1H; 3.66, s, 3H; 3.72, m; 3.72, s, 3H; 3.78, dd, 11.7, 2.4Hz, 1H; 3.86, s, 3H; 3.9, dd, 14, 6.8 Hz, 1H; 4.02, t, 11.2 Hz, 1H;4.12, ddd, 11, 6.8, 2-8 Hz, 1H; 4.28, d, 14 Hz, 1H; 4.60, S, 1H; 5.04,d, 6.8 Hz, 1 H; 5.26, S, 1H; 6.29, d, 2 Hz, 1H; 6.45, d, 2 Hz, 1H; 6.69,brd, 9 Hz, 2H; 6.86, m, 2H; 7.06, m, 2H; 7.06, m, 1H; 7.10, brd, 9 Hz,2H; ¹³C NMR (CDCl₃), (ppm) 50, 52, 55, 55, 55, 55.8, 59.6, 62.5, 67.6,71.4, 79.6, 92.8, 93.4, 94.3, 95.2, 101.8, 109.4, 112.8, 126.2, 126.6,127.5, 127.5, 128.9, 136.6, 157.1, 158.8, 159.8, 160.2, 170.7.
 26. Acomposition comprising a compound of claim 1 together with apharmaceutically acceptable carrier excipient or diluent.
 27. Thecomposition according to claim 26 wherein the compound is of Formula (i)as defined in claim 5 or (ii) as defined in claim
 23. 28. A method forthe treatment of cancer or a cancerous condition wherein the cancer orcancerous condition is selected from the group consisting of leukemia,sarcoma, breast, colon, bladder, pancreatic, endometrial, head and neck,mesothelioma, myeloma, oesophagal/oral, testicular, thyroid, cervical,bone, renal, uterine, prostate, brain, lung, ovarian, skin, liver andbowel and stomach cancers, tumours and melanomas, comprising theadministration of a treatment effective amount of a compound accordingto claim 1 to a subject in need thereof.
 29. The method according toclaim 28 wherein the compound is of Formula (i) as defined in claim 5 or(ii) as defined in claim
 23. 30. A method for the treatment of a diseasestate or condition associated with cellular hyperproliferation whereinthe disease state or condition is selected from the group consisting ofatherosclerosis, restinosis, rheumatoid arthritis, osteoarthritis,inflammatory arthritis, psoriasis, periodontal disease and virallyinduced cellular hyperproliferation, comprising the administration of atreatment effective amount of a compound of claim 1 to a subject in needthereof.
 31. The method according to claim 30 wherein the compound is ofFormula (i) as defined in claim 5 or (ii) as defined in claim
 23. 32. Acomposition comprising the isomer according to claim 24 or 25 togetherwith a pharmaceutically acceptable carrier, excipient or diluent.
 33. Amethod for the treatment of cancer or a cancerous condition wherein thecancer or cancerous condition is selected from the group consisting ofleukemia, sarcoma, breast, colon, bladder, pancreatic, endometrial, headand neck, mesothelioma, myeloma, oesophagal/oral, testicular, thyroid,cervical, bone, renal, uterine, prostate, brain, lung, ovarian, skin,liver and bowel and stomach cancers, tumours and melanomas, comprisingthe administration of a treatment effective amount of the isomeraccording to claim 24 or 25 to a subject in need thereof.
 34. A methodfor the treatment of a disease state or condition associated withcellular hyperproliferation wherein the disease state or condition isselected from the group consisting of atherosclerosis, restinosis,rheumatoid arthritis, osteoarthritis, inflammatory arthritis, psoriasis,peridontal disease and virally induced cellular hyperproliferation,comprising the administration of a treatment effective amount of theisomer a according to claim 24 or 25 to a subject in need thereof.